Sdad assay and uses thereof

ABSTRACT

Methods for screening human sperm using a sperm DNA accelerated decondensation (SDAD) Test, readable at a 5 minute time point, are disclosed. The method is useful in the screening and clinical management for a reproductively challenged human couple. The methods disclosed are highly predictive of ICSI outcome. Methods for screening human sperm using a sperm DNA decondensation (SDD) Test, readable at a 15 minute time point, are disclosed. This test is highly predictive of IUI and IVF outcome. Also dislosed are methods for selecting an appropriate assisted reproductive technique (ART) for a reproductively challenged couple. These methods provide a decision tree that employs a patient&#39;s sperm test results in a sperm DNA accelerated decondensation (SDAD) Test, and sperm DNA decondensation (SDD) Test, wherein an appropriate assisted technique may be selected from intrauterine insemination (IUI), IVF or ICSI. Also, methods are described that will identify males who may benefit from anti-oxidant therapy and/or a varicocelectomy before using their sperm in ART. An automated scoring method is also disclosed for assessing sperm activity after a 5-minute incubation in egg extract (SDAD Test), and after a 15-minute incubation in egg extract (SDD Test). Finally, an automated sperm processing protocol is also disclosed for the rapid preparation of sperm for analysis in both the SDD and SDAD Tests.

PRIORITY CLAIM

This application is a non-provisional of, claims priority to and the benefit of U.S. Provisional Patent Application Ser. No. 60/957,117, filed on Aug. 21, 2007, the entire contents of which are incorporated herein by reference.

BACKGROUND OF INVENTION

The process of fertilization involves a series of events including: a) sperm binding to, and then penetration of the oocyte zona pellucida via the acrosome reaction, b) sperm fusion with the oocyte plasma membrane, c) oocyte activation (release of the cortical granules to prevent multiple sperm from fertilizing the egg), d) sperm activation and pronucleous formation (Abou-Haila A. et al. 2000: Furlong, L. et al. 2005).

Sperm activation initiates post-fertilization upon entry into the ooplasm where the compacted sperm DNA undergoes many alterations as it develops into a male pronucleus. During the first phase of sperm activation or decondensation, the chromatin condensation acquired during spermiogenesis is reversed. Protamines are exchanged with histones reformatting the sperm chromatin allowing for pronucleus formation (Ballhom et al., Wasserman et al.). Once the sperm decondensation is complete, the DNA synthesis phase occurs, that is followed by recondensation of the sperm chromatin in preparation for the cell division that will result in the 2-cell stage embryo (Longo et. al., (1991)).

A need exists in the medical arts for a clinical sperm screening for a protocol that provides assessment of sperm activation events that are not screened. These sperm activation events include: sperm DNA accelerated decondensation (SDAD) and/or sperm earlyDNA synthesis (SEDS). A need continues to exist in the clinical arts for a panel of tests of sperm activation events useful in prescribing the assisted reproductive technology (ART) having the highest probablility for success for a particular patient and/or reproductively challenged couple, and that has the least emotional and financial impact. A need also exists for a test that can identify males determined to have a varicocele(s) who can benefit from a varicocelectomy. Finally, a need exists for protocol(s) that provide a more rapid processing and assessment of sperm analyzed in sperm activation tests that include sperm DNA decondensation.

SUMMARY OF THE INVENTION

In a general sense, a rapid sperm screening assay that is predictive for human sperm reproductive and/or fertilizing capacity is provided in the present disclosure. In some embodiments the assay provides for a highly predictive method for identifying males who produce sperm with low fertilizing capacity, or in the cases where sperm results in a pregnancy, a high probability that the pregnancy will not progress beyond the first trimester, i.e. a low probability of a take home baby. In one aspect, the present invention identifies such males when their sperm is analyzed in a 5 minute to 10 minute in vitro assay that demonstrates a quantifiable difference between a patient's sperm, and the sperm from a known fertile male. In yet other embodiments, this in vitro assay is the sperm DNA accelerated decondensation (SDAD) Test. In other embodiments, the in vitro assay is the sperm early DNA synthesis (SEDS) Test. In yet other embodiments, the in vitro assays include the SDAD Test, SDD Test, and SEDS Test.

In another aspect, a sperm screening method is provided that is more highly predictive of a take-home baby likelihood when using an assisted reproductive technique (ART). By way of example, such as assisted reproductive technique is the intracytoplasmic sperm injection (ICSI). The presently disclosed method provides a predictive value that is highly statistically significant (p<0.01).

In some embodiments, a screening method is provided that is useful in identifying male sperm donors who will have a low probability of successful ART attempts at pregnancy (success being defined as live birth), such as when using ICSI. Other sperm screening assays, such as the sperm DNA decondensation (SDD) Test, the sperm penetration assay (SPA), and the sperm chromatin structure assay (SCSA) have all been found to not be predictive of ICSI live birth outcome. Thus, in some aspect, the present disclosure describes a screening method that can predict ICSI outcome. In some embodiments, this screening method comprises obtaining a semen sample from a potential sperm donor that has been determined to provide a normal sperm assay result in a SPA and SDD Test, and assessing the sample for sperm DNA accelerated decondensation (SDAD) in a frog egg extract as described herein. In some embodiments, the sperm DNA accelerated decondensation (SDAD) is a measure of chromatin DNA decondensation evidenced after about a 5 (five) minute incubation interval in a frog egg extract. In other embodiments, a sperm sample with essentially complete sperm chromatin DNA decondesation at the five (5) minute time interval identifies an unsatisfactory sperm donor for an ICSI pregnancy attempt.

In some embodiments, a screening method is provided for identifying a male sperm donor having a low probability of an successful ART attempts at pregnancy (e.g., IUI and IVF). The screening method has relatively low predictive capacity for determining a couple's success, success being defined as live birth, when sperm from the male partner is used in an ICSI attempt at pregnancy. In some embodiments, the screening method comprises obtaining a semen sample from a potential sperm donor that has been determined to have an abnormal SDD Test score a (delay in DNA decondensation when incubated in frog egg extract). In other embodiments, the delay in sperm DNA decondensation is a measure of chromatin DNA decondensation evidenced after about a 15 (fifteen) minute incubation interval in a frog egg extract. In other embodiments, a sperm sample with an abnormal response in the SDD Test identifies an unsatisfactory sperm donor for an IUI and/or IVF pregnancy attempt. However, because the SDD Test has no predictive capacity for determining if a patient will succeed in an ICSI attempt at pregnancy, such patients can go directly to ICSI attempts at pregnancy.

It is also an object of the invention to provide an automated sperm screening assay having predictive value for assuring pregnancy outcome and/or a successful pregnancy attempt, where natural, or ART attempts at pregnancy have failed, and that this automated approach provides the same predictive value determined by the presently used manual approach that is not amiable to processing and the scoring of large numbers of samples. In other embodiments, the particular sperm screening assay is the SDAD or SDD Tests, or both. In other embodiments, the automated assay is a high throughput sperm screening method that employs a 96-microtiter well plate, each plate comprising a volume of a frog egg extract. Optionally, and in some embodiments, each microtiter well plate may include frog egg extract containing a DNA labeling agent, such as tritiated thymidine triphosphate (when autoradiography is used to analyze DNA synthesis) or 5-Bromo-2′-deoxyuridine (BUdR), when sperm will be stained with a fluorescent tagged anti-BUdR antibody, and the DNA synthesis analyzed using an automated system/image analysis system described herein. Once the extract sperm mixture is incubating, aliquots will be removed at 5 minutes (SDAD Test), and 15 minutes (SDD Test) and the sperm in these aliquots fixed and stained for subsequent analysis as described herein, and scored using an image analysis system. In another embodiment sperm, in an additional aliquot will be analyzed manually using phase contrast microscopy at a 5 minute time point (SDAD Test) and a 15 minute time point (SDD Test), as described herein.

In another embodiment, a method is described for using the SDD Test to identify an unsatisfactory sperm donor whose abnormal score is determined to be related to the patient also having a varicocele(s). When identified, such individuals have been found to benefit from a varicocelectomy.

The following definitions are used throughout the description of the present invention:

As used in the description of the present invention, the term, “hyperdecondensed sperm” is defined as sperm with a 2-fold increase in size over that observed in the fully decondensed sperm. As used in the present description, a “successful” pregnancy is defined as a live birth resulting from a pregnancy achieved using an assisted reproductive technique (ART).

As used in the description of the present invention, the term, “reproductively challenged couple” is defined as a human male and a human female that have been involuntarily infertile for 1 or more years. Approximately forty percent (40%) of these couples are infertile due to male factor(s), forty percent (40%) are infertile due to female factor(s), and 20% are infertile due to combined male and female factors.

As used in the description of the present inventor, the term, “take-home baby” is defined as a live human birth.

The following abbreviations are used throughout the description of the invention:

LPO=lipid peroxidation

SEDS=sperm early DNA synthesis;

HSAA=human sperm activation assay;

SDAD=sperm DNA accelerated decondensation assay;

ROS=reactive oxygen species;

PBS=phosphate buffered saline

ICSI=intracytoplasmic sperm injection

SDD=sperm DNA decondensation

MDA=Malondialdehyde

4-HNE=4-hydroxynonenal

NBT=nitroblue tetrazolium

PMNL=polymorphonuclear leukocytes

ART=assisted reproductive technology

PMA=phorbol-12 myristate-13 acetate

HOST=hypo-osmotic swelling test

WHO=World Health Organization

HHE=4-hydroxy-2hexenal

SDD=sperm DNA decondensation

ROS=reactive oxygen species

SPA=sperm penetration assay

SCI=sperm capacitation index

ODR=outcome delivery rate

OR=odds ratio

PPV=positive predictive value

4-HA=hydroxyalkenals

VX=variocelectomy

SC=spontaneous conceptions

SCSA=sperm chromatin structure assay

SDFA=sperm DNA fragmentation assay

IUI=intrauterine insemination

IVF=in vitro fertilization

H₂O₂=hydrogen peroxide

BUdR=5-Bromo-2′-deoxyuridine

H³-TTP=tritiated thymidine triphosphate

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Lipid peroxidation (LPO) in H₂O₂ treated sperm. LPO was determined using a colorimetric assay for malondialdehyde (MDA) and 4-hydroxyalkenals (Oxford Biochemical Research). Each bar is the mean±SD. Means are significantly different (p<0.001 using ANOVA: 1) when comparing the control untreated sperm with sperm treated with 10, 50 and 100 μM H₂O₂ (black asterisks), and 2) when comparing the lipid peroxidation in each dose at two exposure times (15 min and 1 hr). Black crosses indicate significant differences.

FIGS. 2A-E and 2 a-e: Pictorial representation of the effects of H₂O₂ on human sperm activation. All sperm were photographed using phase contrast microscopy, a 60× objective, and were printed at the same magnification. (2A-E) HSAA normal responses in untreated control sperm incubated in egg extract. (2 a-e) Sperm exposed to 100 μM H₂O₂ for 1 hr and then incubated in egg extract. The arrows point to examples of abnormal sperm activation responses. Bar represents 10 μm. (2A, a) Typical examples of permeabilized sperm before incubation in the egg extract (Time Zero); (2B, b) Sperm decondensation after a 5 min incubation in egg extract with a partially decondensed sperm (2B) and partially and fully decondensed sperm (2 b). (2C, c) Sperm decondensation after a 15 min incubation in egg extract with typical fully decondensed sperm (2C), and hyperdecondensed sperm (c; see white arrows). (2D, d) Examples of recondensed sperm after a 2 hr incubation in the egg extract. (2E, e) Sperm after a 3 hr incubation in egg extract. (2D and 2E) Sperm with normal recondensation after a 2 and 3 hr incubation in egg extract, respectively. (2 d and 2 e) Sperm with abnormal recondensation are shown after a 2 and 3 hr incubation in egg extract, respectively. Note the hyperdecondensed sperm that did not recondense (e) and recondensed sperm with different nuclear sizes and shapes (2 d and 2 e).

FIG. 3A-3B: Effects of hydrogen peroxide dose-time exposure on human sperm activation. Bar is the mean±SD of sperm decondensation after a 5 and 15 min incubation in egg extract. Asterisks indicate significant difference when comparing H₂O₂ treated sperm after a 15 min (3 a and 3 c) and 1 hr (3 b and 3 d) exposure with untreated control sperm using ANOVA. p values <0.05 were considered as significant.

FIG. 4. Effect of ROS dose-time exposure on human sperm activation. Bar is the mean±SD. Sperm recondensation was scored after a 2 and 3 hr incubation in the egg extract. Asterisks indicate significant differences when comparing H₂O₂ treated sperm after 15 min and 1 hr exposures with control untreated sperm using ANOVA, p values <0.05 were considered significant. Since there were no differences in sperm scored after a 2 and 3 hr incubation in the egg extract, these results were combined. Recondensation and hyperdecondensation are significantly different in sperm exposed to 50 and 100 μM H₂O₂ for 15 min (4 a) and 1 hr (4 b).

FIG. 5 a-5 d DNA synthesis in H₂O₂ treated sperm incubated in egg extract for 5 and 15 minutes. ³H-TTP incorporated into sperm DNA were determined in sperm exposed to 0, 10, 50 and 100 μM H₂O₂ for 15 min and 1 hr. ³H-TTP incorporation was scored after a 5 and 15 min incubation in egg extract. Thymidine incorporation into sperm was scored as follows: unlabeled (less than 5 black granules), medium label (from 5 to 25 black granules), and heavy label (more than 25 black granules). Each value is the mean±SD. Means are significantly different when the p values were <0.05 when comparing with control untreated sperm using ANOVA. Asterisks indicate significant differences. (5 a and 5 b) ³H-TTP incorporation after a 5 min incubation in the egg extract. DNA synthesis was observed in sperm exposed to 50 and 100 μM H₂O₂ for 15 min (5 a) and 1 hr (5 b). Irregular medium, and heavy ³H-TTP incorporation were detected in sperm after a 15 min incubation in egg extract in sperm exposed to 50 and 100 μM H₂O₂ for 15 min (5 c) and 1 hr (5 d).

FIG. 6 a-6 d—DNA synthesis in H₂O₂ treated sperm incubated in egg extract for 2 and 3 hr. ³H-TTP incorporation was scored in sperm exposed to 0, 10, 50 and 100 μM H₂O₂ for 15 min and 1 hr. ³H-TTP incorporation was determine after a 2 and 3 hr incubation in the egg extract. After a 2 and 3 hr incubation in the egg extract sperm with increased medium and heavy label was observed in sperm exposed to 50 and 100 μM H₂O₂ for 15 min (6 a and 6 c) and 1 hr (6 b and 6 d). Sperm exposed to 50 and 100 μM H₂O₂ for 1 hr had an abnormal increase in the number of sperm with medium incorporation of ³H-TTP and decrease in the number of sperm with heavy incorporation (6 d).

FIG. 7—Data fields of images captured and analyzed using an image analysis system; FIG. 7 a—Abnormal SDD response: Phase contrast SDD Test Score=44.8: Image Analysis scoring of 356 cells=38.9; Note the microwells etched into the glass that the sperm settles on or in providing for a large number of single sperm (not clumped together as 2 or more cells) that can be quickly and accurately analyzed. When you change fields you must refocus before capturing the images. Hence, the automatic focusing system described in FIG. 7 b—Normal SDD Response: Phase contrast SDD Test Score=94.4: Image analysis scoring of 218 cells=93.9.

FIG. 8—Comparison of SDD Test results obtained when scoring in real time manually using phase contrast microscopy, with results obtained when scoring using an image analysis system (fluorescence microscopy). The 2 scoring approaches provide essentially identical results (correlation coefficient=0.9831)

FIG. 9—Pictorial representation of autoradiographes of control untreated sperm (9A-9E) and sperm exposed to 100 μM H₂O₂ for 1 hr (9 a-e) assayed in the HSAA in the presence of ³H-TTP. All the pictures were photographed using bright microscopy 60× objective. The black bar represents 10 μM, and black arrows point to abnormal sperm activation. All pictures were printed at the same magnification.

(9A-E) Normal responses (control untreated sperm). (9 a-e) Sperm exposed to 100 μM H2O₂ for 1 hr. (9A and a) Sperm at time zero before incubation in the egg extract. (9B and 9 b) Sperm after a 5 min incubation in egg extract. Notice that sperm exposed to 100 μM H₂O₂ for 1 hr had an early initiation of DNA synthesis (9 b). (9C and 9 c) Sperm after a 15 min incubation in the egg extract. Again, notice the early initiation of DNA synthesis in sperm exposed to 100 μM H₂O₂ for 1 hr (9 c). (9D, 9 d and 9E, 9 e) Sperm after a 2 and 3 hr incubation in egg extract, respectively. (9D and 9E) Untreated control sperm nuclei undergo recondensation with regular consistency in size, shape, and no early initiation of DNA synthesis. However, sperm exposed to 100 μM H₂O₂ for 1 hr can be seen to show irregular recondensation, aggregation, differences in size and shape, as well as the presence of sperm arrested in a hyperdecondensed state (9 d and 9 e).

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to methods for assessing sperm fertility through the use of a sperm DNA accelerated decondensation (SDAD) Test in combination with a delayed sperm DNA decondensation (SDD) Test, as well as a method for identifying the most appropriate assisted reproductive technology (ART), thus individualizing the treatment for reproductively challenged couples. In particular embodiments, a short (e.g. 5 minute) time point is employed in the assay for accelerated sperm DNA decondensation assessment, and a 15 minute time point is employed in the assay for delayed DNA decondensation assessment as part of the method.

The invention also provides a high-through put method for clinical sperm assessments, wherein the SDAD and SDD Tests are performed as part of an automated system employing fluorescence microscopy. Most recent data indicates that while still predictive for failure in IUI and IVF attempts at pregnancy, the SDD Test has no predictive value for ICSI presumably due to the way sperm is now prepared for ART that includes a density gradient separation. The SDAD Test does identify a group of patients that will have little chance for success in ICSI ART attempts at pregnancy, and is the only test available that can identify such patients. However, this group of patients are all normal in the SDD Test, so the SDD Test (an abnormal score) can now be used to determine patients who should be directed immediately to ICSI, as this is their most probable and reasonable chance for a successful pregnancy.

The invention also provides a method to identify males who can benefit from a varicocelectomy. Such individuals, upon finding their SDD Test results are abnormal, and having a Urologist find a varicocele(s) will benefit from a varicocelectomy. After the varicocelectomy is performed, the patient will be given 3-6 months to heal. When an improvement using the SDD Test score is found, such individuals now have an improved chance for fathering children by natural conception or ART.

Various modifications and changes can be made to the teachings herein without departing from the spirit and scope of the invention.

The following examples are provided to demonstrate various aspects and methods of the invention, and are provided to enhance the understanding of the methods described herein.

The following examples are in no manner intended to limit the scope of the inventive methods and/or their clinical or other applications.

EXAMPLE 1 Materials and Methods

The present example provides a detailed description of some of the materials and methods employed in the examples that follow.

Materials and Methods:

Chemicals:

Lipid peroxidation of the sperm cellular membrane was determined using the Oxford Biomedical Research Kit Fr 12.

Autoradiography Emulsion, Type NTB2; and photographic developer D-19 by KODAK (Eastman Kodak, Rochester, N.Y.). All the chemicals were purchased from Sigma Chemical Company (St Louis, Mo., USA).

Experimental Design:

In vitro oxidative stress exposure; human sperm from a fertile male were exposed to increasing hydrogen peroxide H₂O₂ concentrations (0, 10, 50 and 100 μM), and incubated at 37° C., for 15 min and 1 hr.

Lipid peroxidation (LPO) measurements and HSAA responses were analyzed in each treatment to determine the effect(s) H₂O₂ concentration- and time exposure on human sperm activation responses.

Hydrogen Peroxide Dose-Response:

Sperm Preparation and Oxidative Treatments:

Semen ejaculate was collected in a sterile jar by masturbation after 3-5 days of sexual abstinence from fertile males who had previously been shown to produce sperm that responded normally in the HSAA. Sperm were isolated from the seminal plasma as previously described in Brown et al., 1995. The sperm pellet was resuspended in phosphate buffered saline (PBS), pH=7.4, and divided into 4 aliquots. Each aliquot (equal sperm concentrations) was incubated at 37° C. in PBS with H₂O₂ concentrations of 0, 10, 50, and 100 μM for 15 min and 1 hr. All aliquots were divided into 2 portions. One portion was used to determine LPO. The remaining portion was centrifuged at 1500 g for 15 min. at room temperature, the sperm in the pellet resuspended and analyzed in the HSAA.

Lipid Peroxidation Measurements:

Aliquots of sperm previously exposed to increasing concentrations of H₂O₂ designed to determine LPO were centrifuged at 10,000 g for 10 min at 4° C., the supernatants placed on ice, and the sperm pellets (approximately 20 million cells) were sonicated using an XL Ultrasonic Cell Disruptor (Microson). Sonication was performed in 3 cycles consisting of 5 sec of sonication followed by 30 sec of incubation on ice. Following sonification, samples were centrifuged at 10,000 g for 10 min. An Oxford Biomedical Research Kit Fr 12 was used to determine LPO (MDA+Hydroxyalkenal concentrations) in the supernatants and the sonicated sperm. This technique is based on the reaction of a chromogenic reagent, N-methyl-2 phenylindole, with MDA and 4-hydroxyalkenals at 45° C. One molecule of either MDA or 4-hydroxyalkenals reacts with 2 molecules of N-methyl-2 phenylindole to produce a stable chromophore that can be quantified at 586 nm.

Human Sperm Activation Assay (HSAA):

The HSAA was performed as described by Brown et al. 1995. Briefly, sperm are washed and permeabilized using lysolecithine.

In a micro-centrifuge tube, the reaction mix was prepared; 8 μl of sperm suspension (200,000 sperm) was added to 100 μLof frog egg extract that contained 8 μl of ³H-TTP (8 μCi). At 5 and 15 min, and 2 and 3 hr incubation times in the egg extract, 5 μl aliquots were taken, wet mounts prepared, and phase contrast microscopy used to determine the percentage of sperm that were fully decondensed and recondensed, respectively, scoring 50 nuclei per slide. At the 5 and 15 min, and 2 and 3 hr time points, 25 μl aliquots of the reaction mix was removed and mixed with 75 μl PBS. Two 50 μl cytopreps were made for each aliquote as described by Brown et al., 1992. The dried cytopreps were fixed in one part acetic acid, and three parts cold methanol, and Giemsa stained for 1 hr followed by a 10 min wash in Giemsa buffer. Slides were examined using light microscopy and analyzed using Metamorph software. Autoradiography was performed using cytoprep slides as described by Brown et al. 1995.

Statistics:

In Vitro study:

The means and standard deviation from (untreated) control sperm was compared with sperm treated with 10-100 μM H₂O₂ at two exposure times (15 min and 1 hr) for: lipid peroxidation values, sperm activation, decondensation at 5 and 15 min, DNA synthesis at 5 and 15 min and 2 and 3 hr, and recondensation at 3 hr. ANOVA (Sigma Stat) analysis of the data was performed, and p values <0.05 were considered as significant.

EXAMPLE 2 Use of the HSAA and the SDAD Test in Identifying Reactive Oxygen Species (ROS) Damage

The present example is provided to demonstrate the utility of the HSAA and the 5-minute time point SDAD Test, an addition to the HSAA, in the identification of damage resulting from exposure to ROS.

The HSAA as described in Example 1 was used in the present study.

Experimental Design:

In vitro oxidative stress exposure; human sperm from a fertile male were exposed to increasing hydrogen peroxide (H₂O₂) concentrations (0, 10, 50 and 100 μM), and incubated at 37° C., for 15 min and 1 hr.

Lipid peroxidation (LPO) measurements and HSAA responses were analyzed in each treatment to determine the effect(s) of H₂O₂ concentration and time exposure on human sperm activation responses.

Hydrogen Peroxide Dose-Response:

Sperm Preparation and Oxidative Treatments—

Semen ejaculate was collected in a sterile jar by masturbation after 3-5 days of sexual abstinence from fertile males who had previously been shown to produce sperm that responded normally in the HSAA. Sperm was isolated from the seminal plasma as previously described in Brown et al. 1995. The sperm pellet was resuspended in phosphate buffered saline (PBS), pH=7.4, and divided into 4 aliquots. Each aliquot (equal sperm concentrations) was incubated at 37° C. in PBS with H₂O₂ concentrations of 0, 10, 50, and 100 μM for 15 min and 1 hr. All aliquots were divided into 2 portions. One portion was used to determine LPO.

The remaining portion was centrifuged at 1500 g for 15 min at room temperature. The pellet was analyzed in the HSAA.

Lipid Peroxidation Measurements:

Aliquots of sperm previously exposed to increasing concentrations of H₂O₂ designed to determine LPO were centrifuged at 10,000 g for 10 min at 4° C., the supernatants were placed on ice, and the sperm pellets (approximately 20 million cells) were sonicated using an XL Ultrasonic Cell Disruptor (Microson). Sonication was performed in 3 cycles consisting of 5 sec of sonication followed by 30 sec of incubation on ice. Following sonification, samples were centrifuged at 10,000 g for 10 min. An Oxford Biomedical Research Kit Fr 12 was used to determine LPO (MDA+Hydroxyalkenal concentrations) in the supernatants and the sonicated sperm. This technique is based on the reaction of a chromogenic reagent, N-methyl-2 phenylindole, with MDA and 4-hydroxyalkenals at 45° C. One molecule of either MDA or 4-hydroxyalkenal reacts with 2 molecules of N-methyl-2 phenylindole to produce a stable chromophore that can be quantified at 586 nm.

1. Cellular Events of Human Sperm Activation:

A. Nuclear Decondensation and Recondensation—

These results indicate that H₂O₂ concentration (50 and 100 μM) and time exposure are the main factors that induced abnormal decondensation responses including: 1) the unique observation of early recondensation, and 2) the novel phenomenon of hyperdecondensation.

B. Nuclear Recondensation—

Sperm chromatin recondensation after a 2 and 3 hr incubation in egg extract (FIG. 4).

Since no differences between 2 and 3 hr incubation times in egg extract were noted, results were combined. In addition, spermatozoa have already advanced beyond decondensation by two and three hours. For this reason, fully decondensed, partially decondensed, and non-decondensed sperm were not observed. No sperm were hyper-decondensed in the control and 10 μM H₂O₂-treated sperm. Hyper-decondensation occurred in 35.2% and 45±2% of the sperm treated with 50 and 100 μM H₂O₂, respectively (15 min exposure, FIG. 4 a). Hyper-decondensed sperm occurred in 30±2% and 45±3% of the sperm treated with 50 and 100 μM H₂O₂, respectively (1 hr exposure, FIG. 4 b). Recondensed sperm were observed in 98±2% of both the control, and 10 μM H₂O₂-treated sperm (15 min and 1 hr exposure). Recondensation occurred in 66±2.3% and 57±2% of the sperm exposed to 50 and 100 μM H₂O₂ respectively (15 min exposure, FIG. 4 a). Recondensation occurred in 70±3% and 54±2% of the sperm exposed to 50 and 100 μM H₂O₂ for 1 hr, respectively (FIG. 4 b).

These results indicate that 1) nuclear recondensation is H₂O₂ concentration and time exposure dependent, and 2) oxidative stress promotes nuclear arrest in the hyperdecondensed state.

C. DNA Synthesis (³H-TTP Incorporation)—

The DNA synthesis was determined by autoradiography as previously described in Materials and Methods.

³H-TTP incorporation into sperm DNA after a 5 min incubation in egg extract (FIGS. 5 a, b and 9 b).

No sperm were positive for ³H-TTP incorporation in the control and 10 μM H₂O₂-treated sperm after 15 min and 1 hr exposures (FIGS. 5 a, b and 9B). Sperm classified as having medium ³H-TTP labeling were observed in 18±3.5% and 36±3% of the 50 μM H₂O₂-treated sperm after a 15 min and 1 hr exposure, respectively. Approximately 25±5% and 48±2% of the 100 μM H₂O₂-treated sperm had medium ³H-TTP labeling after a 15 min and 1 hr exposure, respectively. Sperm heavily labeled by ³H-TTP were observed in 10±3.5% and 28±2% of sperm exposed to 50 μM H₂O₂ and 15±5% and 31±3% of the sperm exposed to 100 μM H₂O₂ for 15 and 1 hr, respectively (FIGS. 5 a, b and 9 b).

³H-TTP incorporation into sperm DNA after a 15 min incubation in egg extract (FIGS. 5 c and 5 d).

Sperm chromatin decondensation and recondensation after a 5 min incubation in egg extract (FIGS. 3 a: 15 min H₂O₂ exposure; and 3 b: 1 hr H₂O₂ exposure). Sperm chromatin decondensation and recondensation after a 15 min incubation in egg extract (FIGS. 3 c: 15 min H₂O₂ exposure; and 3 d: 1 hr H₂O₂ exposure).

Approximately 28±4% of the sperm were fully decondensed in both the control and 10 μM H₂O₂-treated sperm (15 min exposure) and 30±6% at 1 hr exposure. Fully decondensed sperm were observed in 43.3±3.7% and 46.6±6% of the 50 μM H₂O₂ treated sperm after a 15 min and 1 hr exposure, respectively. Fully decondensed sperm were observed in 56.6±4.7% and 64.5±4.5% of the 100 μM H₂O₂-treated sperm after a 15 min and 1 hr exposure, respectively. Approximately 30% of both the control, and 10 μM H₂O₂-treated sperm were partially decondensed (15 min and 1 hr exposures). Approximately 1% of both the 50 and 100 μM H₂O₂-treated sperm (15 min and 1 h exposures) were partially decondensed. Approximately 35-40% of both the control and the 10 μM H₂O₂-treated sperm (15 min and 1 hr exposures) showed no decondensation. Non-decondensed sperm were observed in 15±3 and 18±4% of the sperm treated with 50 μM and 10±2% of sperm treated with 100 μM H₂O₂ (15 min and 1 hr exposures), respectively. No sperm were hyperdecondensed in the control and 10 μM H₂O₂-treated sperm.

Hyperdecondensation occurred in 38.4%, and 26% of the sperm treated with 50 and 100 μM H₂O₂, respectively, after a 15 min exposure. Hyperdecondensed sperm occurred in 31±−4% and 11±4% of the sperm after a 1 hr exposure. No recondensed sperm were observed in control, 10 μM H₂O₂-treated sperm (15 min and 1 hr exposures), and 50 μM H₂O₂-treated sperm for 15 min exposures. Recondensation occurred in 8±2% of the sperm exposed to 100 μM H₂O₂ for 15 min. Recondensation occurred in 5±3% and 15±2% of the sperm exposed to 50 and 100 μM H₂O₂ for 1 hr, respectively.

The oxidative stress that resulted in the above abnormal in vitro sperm activation in the H₂O₂ treated sperm, may be the result of a combined detrimental effect on: 1) the plasma membrane, and 2) DNA integrity. FIG. 8 demonstrates the observed correlation between Phase Contrast (Scoring done manually) and Fluorescence (Scoring done by an image analysis system) analysis of 8 patient semen samples in the SDD Test (X=Fluorescent Score; Y=Phase Contrast Score).

Possibly, the hyperdecondensation effect can be explained by the massive influx of the activation factors contained in the extract, through the leaky membrane. Damage to the plasma membrane, when lipid peroxidation of polyunsaturated fatty acids occurred, results in an increase in the permeability of the membranes. Also, the ‘oxidative chromatin pre-relaxation’ may accelerate sperm nuclear decondensation (Ollero M et al. 2000; Kemal Duru N et al. 2000; Saleh R A et al. 2002). These findings are in agreement with the results of Brown, et al., 1987 who showed that nuclear factors contained in the egg extract regulate sperm decondensation. In an in vitro decondensation kinetics study, sperm was incubated in an active fraction of proteins isolated from the frog egg extract that contained a 70-fold enrichment of partially purified decondensation activation factors. Full decondensation of the sperm nuclei occurred within 5 minutes, and no recondensation was observed, indicating that the recondensation proteins were removed or inactivated during the purification of the decondensation proteins (Brown D B et al. 1987; Brown D B et al. 1991).

Some abnormal sperm decondensation responses are believed to be a result of exposure to reproductive toxicants that directly affect the chromatin such that there is a delay or an enhancement of in vitro decondensation, depending upon the type of exposure. For example, exposure to alkylating agents causes a delayed decondensation (Perreault S et al. 1987 et al. Sawyer D et al. 1995; Sawyer et al. 1998). Exposure to ROS induced an increase in the kinetics of decondensation (accelerated decondensation). Other abnormal responses such as an increase in the recondensation kinetics, may be a result in altered quantities, or enzyme activity of activation factors related to the decondensation/recondensation processes, again due to the damaged membrane (Brown D B et al. 1987; Brown D B et al 1991; Philpott A et al. 1991; Brown et al 1992; Philpott A et al. 1992; Brown D B et al. 1995; Matsumoto K et al. 1999; Sawyer D et al. 2000).

This study supports that high levels of ROS (50 to 100 μM H₂O₂) results in oxidative stress. The oxidative stress results in membrane and DNA damage, translates to sperm DNA accelerated decondensation (SDAD) when such sperm is analyzed at the 5 minute time point.

Results:

1. Lipid Peroxidation Measurements—

To determine the effect of increasing H₂O₂ concentrations on the production of LPO, a spectrophotometric assay was used to measure the amount of MDA and hydroxyalkenals in sperm treated with 0, 10, 50 and 100 μM hydrogen peroxide (H₂O₂) for 15 min and 1 hr. The means were significantly different in 50 and 100 μM H₂O₂-treated sperm (15 min), and 10, 50 and 100 μM H₂O₂-treated sperm (1 hr), when compared to the untreated control sperm (FIG. 1). These results indicate that lipid peroxidation is concentration- and exposure time-dependent.

2. Effect of Oxidative Stress on Human Sperm Activation —

Sperm chromatin decondensation and nuclear recondensation were evaluated in sperm exposed to increasing concentrations of H₂O₂ for 15 min and 1 hr using the HSAA. Sperm were analyzed at different incubation times in the egg extract using the image analysis Methamorph software system. The sperm nuclear activation was scored in 5 categories (FIG. 2): 1) full decondensation: sperm diameter 25-30 μm (FIG. 2C), 2) partial decondensation: sperm diameter <25 μm (FIG. 2B), 3) non-decondensed (FIG. 2A), 4) hyperdecondensed: sperm diameter >(25-30 μm) [FIGS. 2 b and c], and 5) full recondensation (FIGS. 2D and E).

3. Cellular Events of Human Sperm Activation:

A. Nuclear Decondensation and Recondensation

Sperm chromatin decondensation and recondensation after a 5 min incubation in egg extract (FIGS. 3 a: 15 min H₂O₂ exposure; and 3 b: 1 hr H₂O₂ exposure).

Sperm chromatin decondensation after a 5 min incubation in egg extract (FIGS. 3 a and b).

Approximately 28±4% of the sperm were fully decondensed in both the control and 10 μM H₂O₂-treated sperm (15 min exposure) and 30±6% (1 hr exposure). Fully decondensed sperm were observed in 43.3±3.7% and 46.6±6% of the 50 μM H₂O₂ treated sperm after a 15 min and 1 hr exposure, respectively. Fully decondensed sperm were observed in 56.6±4.7% and 64.5±4.5% of the 100 μM H₂O₂ treated sperm after a 15 min and 1 hr exposure, respectively. Approximately 30% of both the control, and 10 μM H₂O₂-treated sperm were partially decondensed (15 min and 1 hr exposures). Approximately 1% of both the 50 and 100 μM H₂O₂-treated sperm (15 min and 1 hr exposures) were partially decondensed. Approximately 35-40% of both the control and the 10 μM H₂O₂-treated sperm (15 min and 1 hr exposures) showed no decondensation. Non-decondensed sperm were observed in 15±3 and 18±4% of the sperm treated with 50 μM H₂O₂ and 10±2% of sperm treated with 100 μM H₂O₂ (15 min and 1 hr exposures), respectively. No sperm were hyperdecondensed in the control and 10 μM H₂O₂-treated sperm. Hyperdecondensation occurred in 38.4%, and 26% of the sperm treated with 50 and 100 μM H₂O₂, respectively, after a 15 min exposure (FIG. 3 a). Hyperdecondensed sperm occurred in 31±4% and 11±4% of the sperm after a 1 hr exposure (FIG. 3 b). No recondensed sperm were observed in control, 10 μM H₂O₂-treated sperm (15 min and 1 hr exposures), and 50 μM H₂O₂-treated sperm for 15 min exposure (FIG. 3 a). Recondensation occurred in 8±2% of the sperm exposed to 100 μM H₂O₂ for 15 min (FIG. 3 a). Recondensation occurred in 5±3% and 15±2% of the sperm exposed to 50 and 100 μM H₂O₂ for 1 hr (FIG. 3 b).

Sperm Chromatin Decondensation and Recondensation after a 15 Min Incubation in Egg Extract (FIGS. 3 c: 15 min H₂O₂ exposure; and 3 d: 1 hr H₂O₂ exposure).

Approximately 94% of the sperm were fully decondensed in both the control and 10 μM H₂O₂-treated sperm (15 min and 1 hr exposures). Fully decondensed sperm were observed in 53±3% and 58±2% of the 50 μM H₂O₂-treated sperm after a 15 min and 1 hr exposure, respectively. Fully decondensed sperm were observed in 48±1.5% and 47±2% of the 100 μM H₂O₂-treated sperm after a 15 min and 1 hr exposure, respectively. Approximately 5% of the sperm were non-decondensed in the control and the 10 μM treatment at both time points. However, no sperm were non-decondensed for any other concentrations at any of the time points. No sperm were hyperdecondensed in the control and 10 μM H₂O₂-treated sperm. Hyperdecondensation occurred in 45.2% and 37% of the sperm treated with 50 and 100 μM H₂O₂, respectively (15 min exposure, FIG. 3 c). Hyperdecondensed sperm occurred in 30±2% and 33±3.5% of the sperm (1 hr exposure, FIG. 3 d). No recondensed sperm were observed in control, 10 and 50 μM H₂O₂-treated sperm (15 min exposure, FIG. 3 c). Recondensation occurred in 15±3% of the sperm exposed to 100 μM H₂O₂ (15 min exposure, FIG. 3 c). Recondensation occurred in 10±2% and 20±2% of the sperm exposed to 50 and 100 μM H₂O₂ for 1 hr (FIG. 3 d).

No sperm were positive for ³H-TTP incorporation in the control and 10 μM H₂O₂-treated sperm after 15 min and 1 hr exposures (FIGS. 5 c and d). Sperm having medium labeling were observed in approximately 20±3% and 30±10% of sperm exposed to 50 and 100 μM H₂O₂ for 15 minutes, respectively. Approximately 21±5%, 37±9% of the sperm exposed for 1 hr to 50 and 100 μM H₂O₂, had medium labeling, respectively. Sperm heavily labeled by ³H-TTP incorporation were observed in 10±2% and 17.5±7.5% of sperm exposed to 50 μM H₂O₂ and 50±5% and 45±9% of sperm exposed to 100 μM H₂O₂ at 15 min and 1 hr, respectively (FIGS. 5 c and d).

³H-TTP incorporation into sperm DNA after a 2 hr incubation in egg extract (FIGS. 6 a and b).

Sperm with medium labeling were observed in approximately 14.5±4% and 43.5±5% of sperm exposed to 50 and 100 μM H₂O₂ for 15 min and 1 hr (FIGS. 6 a and 6 b), respectively. Approximately 36±2% and 28.5±2.5% of the sperm exposed for 1 hr to 50 and 100 μM H₂O₂, had medium labeling, respectively. Sperm heavily labeled by ³H-TTP incorporation were found in 99±1% of the sperm in the control and 10 μM H₂O₂-treatments at both 15 min and 1 hr exposures (FIGS. 6 a and 6 b). Sperm with heavy labeling were observed in 85±5% and 56.5±3% of the sperm exposed to 50 μM H₂O₂ for 15 min and 1 hr, respectively. Approximately 64±2% and 71±3% of the sperm exposed to 100 μM H₂O₂ for 15 min and 1 hr, showed heavy labeling, respectively (FIG. 9 d).

³H-TTP incorporation into sperm DNA after a 3 hr incubation in egg extract (FIGS. 6 c and 6 d). Sperm with medium labeling were observed in approximately 16.5±2% and 14.5±3% of sperm exposed to 50 and 100 μM H₂O₂ for 15 minutes (FIG. 6 c), respectively. Approximately 25±3% and 40±4% of the sperm exposed for 1 hr to 50 and 100 μM H₂O₂, had medium labeling respectively (FIG. 6 d). Heavily labeled sperm were found in 98±2% of the control and 10 μM H₂O₂-treated sperm after 15 min and 1 hr exposures (FIGS. 6 c and 6 d). Sperm with heavy labeling were observed in approximately 78±6% of the sperm exposed to 50 μM H₂O₂ and 85±5% of the sperm exposed to 100 μM H₂O₂ for 15 min (FIG. 6 c). Approximately 75±3% of the sperm exposed to 50 μM H₂O₂ and 60±4% of the sperm exposed to 100 μM H₂O₂ for 1 hour, had heavy labeling (FIG. 6 d).

These DNA synthesis results indicate that: 1) oxidative stress induces early DNA synthesis (³H-TTP-incorporation) during nuclear decondensation after a 5 and 15 min incubation in the frog egg extract, 2) early DNA synthesis was H₂O₂ concentration and exposure time dependent, 3) abnormal DNA synthesis was observed in the 50 and 100 μM H₂O₂ treated sperm after a 2 and 3 hr incubation in the egg extract, such treated sperm had a significant decrease in heavily labeled nuclei that was again concentration and exposure time dependent.

The results obtained in this study demonstrate that the concentration of hydrogen peroxide and exposure time were the main factors that induced lipid peroxidation that correlated with abnormal sperm activation responses. Abnormal responses included: a) decondensation, b) an increased number of sperm with premature, fully decondensed nuclei, c) the unique observation of early recondensation, and the novel phenomenon of hyperdecondensation after a 5 and 15 min incubation in egg extract. Oxidative stress promoted abnormal recondensation and nuclear arrest in the hyperdecondensed state in 33-42% of the sperm nuclei observed in 50 and 100 μM H₂O₂-treated sperm after a 3 hr incubation in the egg extract. Oxidative stress induced early DNA synthesis activity during nuclear decondensation at 5 and 15 min. The DNA synthesis activity was ROS concentration- and exposure time-dependent.

The oxidative stress that resulted in the above abnormal in vitro sperm activation in the H₂O₂ treated sperm, is a combined detrimental effect on: 1) the plasma membrane, and 2) DNA integrity. FIG. 8 demonstrates the observed correlation between Phase Contrast (Scoring done manually) and Fluorescence (Scoring done by an image analysis system with auto focus) analysis of 8 patient semen samples in th SDD Test. (X=Fluorescent Score; Y=Phase Contrast score). This demonstrates that both scoring methods yield essentially the same results.

Possibly, the hyperdecondensation effect can be explained by the massive influx of the activation factors contained in the extract, through the leaky membrane. Also, the ‘oxidative chromatin pre-relaxation may accelerate sperm nuclear decondensation (Ollero M et al. (2000)); Kemal Duru N et al. (2000); Saleh R A et al. (2002).

In an in vitro decondensation kinetics study, sperm were incubated in an active fraction of proteins isolated from the frog egg extract that contained a 70-fold enrichment of partially purified decondensation activation factors. Full decondensation occurred within 5 minutes, and no recondensation was observed, indicating that the resondensation proteins were removed or inactivated during the purification of the decondensation proteins (Brown D B et al. (1987); Brown D B et al. (1991)).

Some abnormal sperm decondensation responses are believed to be a result of exposure to reproductive toxicants that directly affect the chromatin such that there is a delay or an enhancement of in vitro decondensation, depending upon the type of exposure. For example, exposure to alkylating agents causes a delayed decondensation (Perreault S et al. (1987), Sawyer D et al. (1995); Sawyer et al. (1998)), while exposure to ROS induced an increase in the kinetics of decondensation. Other abnormal responses, such as an increase in the recondensation kinetics, may be a result in altered quantities, or enzyme activity of activation factors related to the decondensation/recondensation processes, again due to the damaged membrane (Brown D B et al. (1987); Brown D B et al. (1991); Philpott A et al. (1991); Brown et al. (1992); Philpott A et al. (1992); Brown D B et al (1995); Matsumoto K et al. (1999); Sawyer D et al. (2000)).

The autoradiography results in the present study indicate that both the control (untreated) and sperm exposed to 10 μM H₂O₂ for 15 min and 1 hr had no incorporation of ³H-TTP after 5 and 15 ml incubations in the egg extract, and normal ³H-TTP incorporation after 2 and 3 hr incubations in frog egg extract. These results are in agreement with previous in vitro studies, which have defined the standard values of ³H-TTP incorporation in fertile males that responded normally in the HSAA (Perreault S et al. (1987); Brown D B et al. (1992); Sawyer et al. (1995); Brown D B; et al. (987), Brown D B et al. (1995); Sawyer D et al. (1998); Sawyer D et al. (2000)). In agreement with the previous studies, a) No ³H-TTP incorporation “unlabeled” during sperm chromatin decondensation was observed at the 5 and 15 min in egg extract, and b) approximately 97 to 99% of the sperm population incorporated ³H-TTP (“heavy label”; more than 25 black granules per sperm nucleus) after 2 and/or 3 hr incubation in the egg extract. However, the ³H-TTP incorporation results of sperm exposed to 50 and 100 μM H₂O₂ for 15 min and 1 hr, show that sperm had abnormal ³H-TTP incorporation including: a) early DNA synthesis after a 5 and 15 min incubation in the egg extract, and b) increase in the number of sperm positive for medium label after 2 and 3 hr incubations in the egg extract. The abnormal ³H-TTP incorporation was H₂O₂ concentration and exposure time dependent.

In vitro studies indicate that chromatin decondensation is required for DNA synthesis, specifically for the formation and maturation of the male pronucleus. DNA synthesis is engineered totally by factors present in the egg extract; one of these factors is the enzyme DNA polymerase α.

In the untreated control sperm, and sperm exposed to 10 μM H₂O₂ for 15 min and 1 hr, no ³H-TPP incorporation was observed. After a 1 hr incubation in egg extract, all sperm are synthesizing DNA. This DNA synthesis is believed to be semi-conservative DNA synthesis typical for S phase of the cell cycle (Brown D B et al. (1987)), Mazia D (1963)).

The early DNA synthesis activity observed in sperm exposed to 50 and 100 μM H₂O₂ for 15 min and 1 hr suggests that the DNA synthesis observed is repair DNA synthesis of the ROS damaged DNA. As sperm does not have DNA repair capacity (Ramos et al. (2001)), the DNA repair activity must be provided by egg cytoplasm. DNA repair activity has been found to be dependent upon oocyte cytoplasmic factors (Baarends et al. (2001)); Ochsendorft (1999)).

The early DNA synthesis is believed to be DNA repair. These results are in agreement with Sawyer at al. (1995). The results of sperm exposed to hydroxylamine, a mono-functional DNA damaging agent, that were subsequently analyzed in the HSAA, indicate that the mutagen induces abnormal sperm activation responses that include minimal decondensation, and the early initiation of DNA synthesis. In this study, it is hypothesized that the damaged DNA, activated DNA repair machinery present in the frog egg extract, and thus resulted in an early onset of DNA synthesis (Zhi et al. (1997); Sawyer D et al. (2000)).

Since sperm does not have DNA repair capacity (Ramos L et al. (2001), the DNA repair activity must be provided by egg cytoplasm. DNA repair activity has been demonstrated in several mouse models for male infertility, and in biopsies taken from infertile males in which DNA integrity is compromised suggests that the repair of DNA-oxidative damage occurs between sperm decondensation and pronucleus formation, and that the DNA repair is dependent on oocyte cytoplasmic factors (Baarends et al., (2001), Ochsendorft F R (1999)). The autoradiography results in this present study are in agreement with the above observations.

The results indicate that H₂O₂ at 50 and 100 μM concentrations produced DNA damage that resulted in abnormal activation responses. Additional studies indicate that sperm exposed to endogenous ROS, or direct exposure to H₂O₂, resulted in a dose-dependent induction of high rates of DNA fragmentation. Such induction was significant at 200 μM concentrations of H₂O₂. The resulting DNA fragmentation was observed using the comet assay (Kemal Duru N et al. (2000)).

The base excision DNA repair pathway is used to repair DNA oxidative damage. In addition, DNA repair mechanisms have been involved not only in the repair of DNA damage in developing germ line cells, but also to enhance specialized gene expression during mammalian gametogenesis (Baarends W et al. (2001)).

This study supports that high levels of ROS (50 to 100 μM H₂O₂) results in oxidative stress. The oxidative stress results in membrane and DNA damage that translates to abnormal responses when such sperm is analyzed in the HSAA.

EXAMPLE 3 Sperm DNA Decondensation (SDD) and Sperm Penetration Assay (SPA) with Gradient Preparation Are Not Predictive of Pregnancy Outcome Using In Vitro Fertilization (IVF) with Intracytoplasmic Sperm Injection (ICSI) Cycles

Several sperm structure tests that detect compromised DNA integrity have been shown to have poor predictive capacity for successful ICSI cycles. This is believed to be the result of improved selection of the best sperm for use in ICSI, by use of a density gradient. Two different sperm function tests, the SDD Test and the SPA, are examined for capacity to predict success in ICSI cycles.

The SDD Test measures the fraction of cells fully decondensed after a 15 minute incubation in frog egg extract, measuring delayed decondensation, while the SPA measures the sperm capacitation index (SCI), the average number of sperm penetrations per zona-free hamster oocyte. The objective of this study was to review the pregnancy outcome in ICSI cycles in relation to the SPA and the SDD Test results using the best sperm looking at SCI improvement over SCI from semen sperm, and semen sperm looking at delayed DNA decondensation in comparison to that observed for sperm from a fertile male, and determine if either of the 2 parameters being evaluated have predictive capacity for determining success in ART.

A prospective, blinded, single center, cohort study was conducted. Outcome was evaluated by delivery rate (ODR), defined as the number of ongoing pregnancies and/or deliveries per ICSI cycle.

Materials and Methods:

Ejaculates were divided into 3 portions, one used for the SPA, the second for the SDD Test and the third frozen for use in a future ICSI cycle. The data consisted of 53 ICSI cycles. Cycles were grouped according to abnormal v normal score in the SPA (<14v≧14 SCI) and the SDD (<80% v≧80%). A gradient preparation was used for sperm preparation for the SPA as well as ICSI. Standard methods were used for ovarian stimulation and embryo culture. Odds ratios (OR) and positive predictive value (PPV) for failure were calculated, and the statistical significance was assessed by the Chi² distribution.

Results:

The SDD is not predictive of ODR in IVF with ICSI cycles.

The SPA is not predictive of ODR in IVF with ICSI cycles.

TABLE 1 SPA SCI(N = 55) SDD (N = 55) Post-gradient <80 ≧80 <14 ≧14 ICSI cycles 21 32 19 36 Delivery rate 42.9% 40.6% 47.4% 41.7% (ODR) OR 1.2 0.79 PPV 57% 52.6% P NS NS

Test: The combined use of the SPA and SDD Test will provide a powerful tool in decision making during the male infertility evaluation and for better prediction of outcome of ICSI attempts at pregnancy involving infertile men, for male infertility. This incorporates two sub-objectives as follows:

Study Protocol:

Design—A prospective blinded study.

The males were from couples where the male partners are admitted for andrology evaluation including the SPA in preparation for IVF. Measurement will be attempted on the very same ejaculates provided for ICSI attempts at pregnancy in IVF.

Statistics for success rates when performing ICSI:

70% Fertilization Rate

45-50% Pregnancy Rate

40% Live Birth Rate

Patients:

Male Age 25-45. Female age: 25-40. Male partners of infertile couples who fulfill the inclusion and exclusion criteria will be included.

Study groups whose samples will be analyzed in both the SDD Test and SPA, as well as ART attempts at pregnancy:

Study Group:

Infertile males from infertile couples for whom SPA would have been prescribed, either leading to IVF treatment or as part of IVF pre-evaluation. This group will be divided into 3 subgroups based on the results of the SPA:

Highest SCI >6,

Highest SCI 1.0-6,

Highest SCI <1.0.

Inclusion Criteria:

Female partner must be checked for the following:

Infection with microorganisms (viral, bacterial or fungal such as Mycoplasma/Ureaplasma, Chlamydia trachomatis, Bacterial Vaginosis) known to be associated with female infertility (Past or Current).

Autoimmune disease (lupus, RA, MS, Diabetes, Hashimoto, etc.) determined while interviewing the infertile couple.

Inflammatory/autoimmune/coagulation blood feature abnormalities as assessed by blood work including tests for Lupus anti coagulant (LAC), Anti cardiolipin Antibodies (ACA), Anti phospholipid antibodies (APA), Natural Killer Cells (NK), Reproductive immunophenotyping (RIP), Anti Microsomal Antibodies (thyroid marker) (AMA) and Factor V (coagulation).

Blood for workup for the female will be sent up to 6 weeks prior to beginning the cycle. One plasma is to be spun and separated for Factor V (sent at ambient temperature) and LAC (sent at ambient temperature), one heparinized whole blood for cellular immunology —NK and RIP (sent ambient temp) and one test-tube with serum spun and separated for ACA, APA and AMA (sent at ambient temperature).

If 10 or more women have one or more of the above problems, these couples will be included and treated for all statistical analyses as a separate group. Otherwise, they will be dropped from the study.

Oligo or normospermic male with >5 million total motile sperm.

Exclusion Criteria:

Age: Patients/donors younger than 25 yr or older than 45 yr.

Patient Recruitment:

The prospective study will enroll 60 patients who fulfill the selection criteria.

Main Outcome Measures:

Diagnostic:

For each patient, the following will be measured:

Standard andrology work-up including concentration of sperm, morphology of sperm, volume of ejaculate, and motility of the sperm in all specimens used for the study. Sperm DNA decondensation in the SDD Test (15 minutes).

Sperm Penetration Assay (SPA) Scores.

Fertilization rates, clinical pregnancy rates, successful pregnancies as defined by any pregnancy continuing past 13 weeks, live births, and the child assessment results at birth.

Pregnancy complications, and gestation week if ended.

Details of the Protocol:

The study will determine the relationship of SDD and/or SPA results and pregnancy results with ART cycles with ICSI.

Semen Samples:

60 patients will be participating in this study. Specimens will be allowed to liquefy by incubating up to 1 hr at room temperature (not less than 22° C.). Specimens will be evaluated on-site in the andrology lab for basic parameters, and then will be prepared as determined from the initial evaluation of the correlation of the sperm preparation in the preliminary study. This will be used in the SDD Test, the SPA, and ICSI attempts at pregnancy.

The Study Protocol:

The sperm from the 60 patients will be split into 3 aliquots:

Aliquot A: SPA—2 million cells. The test will be performed.

Aliquot B: SDD Test—2 million cells will be tested.

Aliquot(s) C: These aliquots will be cryopreserved for future use in ICSI attempts at pregnancy.

All samples for analysis will be blinded.

Only the patient's initials and date of birth information will be provided for tracing and recording purposes. None of the other andrology test results will be provided at the time of test submission. The study will be unblinded for each patient 13 weeks after each patient's sample is used in an ICSI attempt at pregnancy when an on-going pregnancy occurs. If the ICSI attempt at pregnancy is negative, a biochemical pregnancy, or a pregnancy that spontaneously aborts during the first trimester, such patients will be unblinded when either no pregnancy occurs, or the patient spontaneously aborts the fetus. The patient's pregnancy outcomes and child assessment results will be disclosed as soon as the results are available; results to be used in the comprehensive statistical analysis that will be done.

For the SDD Test: Specimen aliquots will be kept in the refrigerator at 2-8° C., until shipped cold by packing with a coolpak. Specimens must be sent no later than 9 days after sample collection. The samples will be analyzed in the SDD Test within 14 days of sample collection. Any sample arriving such that it cannot be analyzed within the 14 day QC window will be rejected, and another sample requested.

The SPA:

The SPA test will be performed as a modified version to Johnson et al. (1990) as described below.

Semen Preparation:

Semen will be washed and concentrated by gradient method. After gradient preparation the sperm will be resuspended in sperm wash medium and diluted 1:1 with Test Yolk Buffer (TYB). The sperm/TYB mixture will then be slowly cooled to 2-8° C. and stored at this temperature for 2-3 days. After this time, sperm wash medium at 37° C. will be added to the cold sperm/TYB mixture, providing a thermal shock. After the sperm/TYB has incubated for 30 minutes at 37° C., the mixture will be centrifuged for 10 min at 600 g. The supernatant will then be removed, up to 1.0 mL sperm wash medium added, and the sperm allowed to incubate for 60 minutes at 37° C. The sample will then be adjusted to a concentration of 5 million total motile sperm/mL.

Controls:

In each assay, one negative and one positive (as previously determined) control will be run in parallel to the study samples.

Ova Preparation:

Frozen hamster ova will be utilized for the SPA. Straws containing ova will be thawed at RT for 2 min in a horizontal position. The straws are then shaken at the crimped end to vigorously mix the sucrose column with ova. The straws will then be incubated, cotton end down, in a 37° C. water bath for 3 min followed by 3 min at RT, cotton end up. Using a pushrod, the ova are then dispensed into a 35 mm culture dish and washed twice in sperm wash medium. The ova will be transferred to fresh sperm wash medium to rest for 10 min at RT. The ova will be washed three times in trypsin (1%) and incubated at RT in the 3rd trypsin drop until the zona is almost depleted (<5 min). The ova are then washed twice in sperm wash medium. The ova will be transferred to fresh sperm wash medium to rest for 5 min at RT.

Sperm/Ova Incubation and Scoring:

Six-seven ova will be placed into each of two 100 μL drops of sperm wash medium covered with oil containing 250,000 total motile sperm and allowed to incubate for 3.5 hr at 37° C. After incubation, the ova will be washed in sperm wash medium to dislodge any loosely bound sperm. The ova will then be placed on a microscope slide with cover slip applied so to flatten the ova for penetration assessment. The number of penetrations (clear zone with discernible tail) will be counted and the Sperm Capacitation Index (SCI) calculated by dividing the total number of penetrations by the total number of ova scored.

The SDD Test:

The SDD test will be performed as described by Brown et al. (1992, 1995).

Egg Extracts Female Xenopus Laevis Oocyte positive frogs are maintained in Repromedix and the frog egg extracts is being produced every six weeks. The extracts are immediately snap deep-frozen in liquid N₂ (LN) for storage. This frog egg extract will be used to induce the sperm DNA decondensation, mimicking the event after sperm entry into the oocyte.

The test: About two million sperm per sample will be washed and permeablized. After four extensive washes with special buffers, the sperm will be treated with dithiotreitol (DTT)-containing buffer. The treated sperm will then be incubated with frog egg extract to induce sperm DNA decondensation. At 15 min 50-100 sperm (the number of sperm that can be score in 5 min) from a fertile male will be scored, and then this process will be repeated for each patient using phase-contract microscopy. The percentage of sperm undergoing full decondensation is recorded. The raw data will be normalized with negative control decondensation value yielding the reportable value as % of the control.

Controls: In each test one negative control and one positive control will be run in parallel with the patient samples. The control specimen is a normal, SDD pre-tested specimen from a sperm bank, or a specimen obtained from an individual who has produced 4 or more ejaculates containing sperm that respond normally at the 15 min time point. This control serves as negative control when used in the complete SDD Test protocol, Patient positive controls that have been identified as being abnormal in routine testing of patient samples sent to Repromedix for analysis in the SDD Test are frozen and used as the positive control.

Outcome Evaluation:

All the results will be statistically evaluated.

Correlation between the SDD Test and SPA scores (linear correlation).

Correlation between the SDD Test and the pregnancy success in the first (any) treatment selected.

Correlation between the SDD Test, and successful pregnancy completion—evaluation of the cut off value for the SDD test that best predicts pregnancy failure, statistical PPV, NPV, specificity, sensitivity and accuracy.

Correlation between each of the two test values and the improvement in quality of all embryos in the respective cycle.

Comparison between the correlations of SDD Test and pregnancy results obtained within the same specimen used for ART v those performed on a separate specimen.

Association between the SDD Test score and environmental/drug/smoking exposures.

Construction of an improved decision-making paradigm involving the two tests including the semen analysis data.

Statistical Analysis:

ANOVA, single factor without replication method and paired t tests (for consequential relations), will be used to compare each of the two test results (SDD, SPA) between patients with successful pregnancy and those with failed pregnancy. P value of <0.05 will be considered statistically significant. If data distribution deviates from normal distribution, or if the variances of the groups under analysis are dramatically different, rank tests (Mann Whitney U test) would be performed. All data will be presented as mean±SEM.

Determination of the best cutoff value predicting pregnancy failure or specific pregnancy outcomes will be done by ROC analysis. Specificity, Sensitivity, PPV and NPV as well as Accuracy will be evaluated using the selected cutoff values.

In such as paired analysis will be performed.

In this study, neither the SPA or the SDD Test have predictive capacity in determining patients' who produce sperm that will fail if used in an ICSI attempt at pregnancy (See Table 1).

EXAMPLE 4 Sperm DNA Accelerated Decondensation (SDAD) Test Scores can be Used as a Predictive of Pregnancy Outcome In Vitro Fertilization (IVF) with Intracytoplasmic Sperm Injection (ICSI) Cycles

Several sperm structure tests that detect compromised DNA integrity have been shown to have poor predictive capacity for successful ICSI cycles. This is believed to be the result of improved selection of the ‘best’ or ‘prime’, sperm for use in ICSI, by use of a density gradient. Here it is determined using the SDAD Test that scores from this test have the capacity to predict failure in ICSI cycles. The SDAD Test measures the fraction of cells fully decondensed after a 5 minute incubation in frog egg extract, measuring accelerated decondensation. The objective of this study was to review the pregnancy outcome in ICSI cycles in relation to the SDAD Test results and determine if the SDAD Test has predictive capacity for determining failure in ART.

Results:

A prospective, double blinded, single center, cohort study.

Outcome was evaluated by delivery rate (ODR), defined as the number of ongoing pregnancies and/or deliveries per ICSI cycle.

Materials and Methods:

The same ejaculates used to perform the SDD Test and the SPA as described in Example 3 that were divided into 3 portions, one used for the SPA, the second for the SDD Test and the third frozen for use in a future ICSI cycle were also used in performing the SDAD Test. However, in addition to scoring for delayed DNA decondensation at 15 minutes, accelerated DNA decondensation was scored by scoring a 5 minute time point from the same egg extract/sperm incubation mixture used in Example 3 to score the 15 minute SDD Test. The data consisted of 53 ICSI cycles. Cycles were grouped according to abnormal v. normal scores in the SDAD Test (>120% v.≦120%). Standard methods were used for ovarian stimulation and embryo culture. Odds ratios (OR) and positive predictive value (PPV) for failure were calculated, and the statistical significance was assessed by Chi² distribution.

TABLE 2 SDAD (N = 55) <120 ≧120 ICSI cycles 42 13 Delivery 54.8% 7.7% rate (ODR) OR 14.5 PPV 92.3% p P < 0.01 Utility Highly predictive of ICSI failure

Test:

Blinded study. The males were from couples where the male partners are admitted for andrology evaluation including the SPA in preparation for IVF. Measurements were performed using sperm from the same ejaculate used for each couple's ICSI attempt at pregnancy.

Statistics for Success Rates when Performing ICSI:

70% Fertilization Rate

45-50% Pregnancy Rate

40% Live Birth Rate

Patients:

Male Age 25-45. Female age: 25-40. Male partners of infertile couples who fulfill the inclusion and exclusion criteria will be included.

Study groups whose samples were analyzed in both the SDD Test and SPA (Example 3), and in the SDAD Test (this example), as well as ART attempts at pregnancy:

Study Group:

Infertile males from infertile couples for whom SPA would have been prescribed, either leading to IVF treatment or as part of IVF pre-evaluation. This group will be divided into 3 subgroups based on the results of the SPA:

Highest SCI >6,

Highest SCI 1.0-6,

Highest SCI <1.0.

Inclusion Criteria:

Female partner must be checked for the following:

Infection with microorganisms (viral, bacterial or fungal such as Mycoplasma/Ureaplasma, Chlamydia trachomatis, Bacterial Vaginosis) known to be associated with female infertility (Past or Current).

Autoimmune disease (lupus, RA, MS, Diabetes, Hashimoto, etc.) determined while interviewing the infertile couple.

Inflammatory/autoimmune/coagulation blood feature abnormalities as assessed by blood work including tests for Lupus anti coagulant (LAC), Anti cardiolipin Antibodies (ACA), Anti phospholipid antibodies (APA), Natural Killer Cells (NK), Reproductive immunophenotyping (RIP), Anti Microsomal Antibodies (thyroid marker) (AMA) and Factor V (coagulation).

Blood for workup for the female will be sent up to 6 weeks prior to beginning the cycle. One plasma is to be spun and separated for Factor V (sent at ambient temperature) and LAC (sent at ambient temperature), one heparinized whole blood for cellular immunology —NK and RIP (sent ambient temp) and one test-tube with serum spun and separated for ACA, APA and AMA (sent at ambient temperature).

If 10 or more women have one or more of the above problems, these couples will be included and treated for all statistical analyses as a separate group. Otherwise, they will be dropped from the study.

Oligo or normospermic male with >5 million total motile sperm.

Exclusion Criteria:

Age: Patients/donors younger than 25 y or older than 45 years.

Control Group:

Healthy fertile semen donors used as controls in the SPA.

Patient Recruitment:

The prospective study will enroll 60 patients who fulfill the selection criteria.

Main Outcome Measures:

Diagnostic:

For each patient, the following will be measured:

Standard andrology work-up including concentration of sperm, morphology of sperm, volume of ejaculate, and motility of the sperm in all specimens used for the study.

Sperm DNA accelerated decondensation in the SDAD Test (5 min).

Fertilization rates, clinical pregnancy rates, successful pregnancies as defined by any pregnancy continuing past 13 weeks, live births, and the child assessment results at birth.

Pregnancy complications, and gestation week if ended.

Details of the Protocol:

Semen Samples:

60 patients will be participating in this study. Specimens will be allowed to liquefy by incubating up to 1 hr at room temperature (not less than 22° C.). Specimens will be evaluated on-site in the andrology lab for basic parameters, and then will be prepared as determined from the initial evaluation of the correlation of the sperm preparation in the preliminary study. This will be used in the SDAD Test, and ICSI attempts at pregnancy.

The Protocol:

The sperm from the 60 patients will be split into 3 aliquots:

Aliquot A: SPA—2 million cells. The test will be performed.

Aliquot B: SDAD and SDD Test—2 million cells will be tested.

Aliquot(s) C: These aliquots will be cryopreserved for future use in ICSI attempts at pregnancy.

All samples for analysis will be blinded.

Only the patient's initials and date of birth information will be provided for tracing and recording purposes. None of the other andrology test results will be provided at the time of test submission. The study will be unblinded for each patient 13 weeks after each patient's sample is used in an ICSI attempt at pregnancy. The patient's pregnancy outcomes and child assessment results will be disclosed as soon as the results are available; results to be used in the comprehensive statistical analysis that will be done.

For the SDAD Test: Specimen aliquots will be kept in the refrigerator at 2-8° C., until shipped cold by packing with a coolpak. Specimens must be sent no later than 9 days after sample collection. The samples will be analyzed in the SDAD Test within 14 days of sample collection. Any sample arriving such that it cannot be analyzed within the 14 day QC window will be rejected, and another sample requested.

Semen Preparation:

Semen will be washed and concentrated by gradient method. After gradient preparation the sperm will be resuspended in sperm wash medium and diluted 1:1 with Test Yolk Buffer (TYB). The sperm/TYB mixture will then be slowly cooled to 2-8° C. and stored at this temperature for 2-3 days. After this time, sperm wash medium at 37° C. will be added to the cold sperm/TYB mixture, providing a thermal shock. After the sperm/TYB has incubated for 30 min at 37° C., the mixture will be centrifuged for 10 min at 600 g. The supernatant will then be removed, up to 1.0 mL sperm wash medium added, and the sperm allowed to incubate for 60 min at 37° C. The sample will then be adjusted to a concentration of 5 million total motile sperm/mL.

Controls:

In each assay, one negative and one positive (as previously determined) control will be run in parallel to the study samples.

The SDAD Test:

The SDAD Test will be performed as described by Brown et al. (1992, 1995) with the new addition of the 5 minute time point to score for accelerated DNA decondensation.

Egg Extracts:

Female Xenopus Laevis Oocyte positive frogs are maintained in Repromedix and the frog egg extracts is being produced every six weeks. The extracts are immediately snap deep-frozen in liquid N₂ (LN) for storage. This frog egg extract will be used to induce the sperm DNA decondensation, mimicking the event after sperm entry into the oocyte.

The Test:

About two million sperm per sample will be washed and permeablized. After four extensive washes with special buffers, the sperm will be treated with dithiotreitol (DTT)-containing buffer. The treated sperm will then be incubated with frog egg extract to induce sperm DNA decondensation. At 5 min 50-100 sperm (the number of sperm that can be score in 5 min) from a fertile male will be scored, and then this process will be repeated for each patient using phase-contract microscopy. The percentage of sperm undergoing full decondensation is recorded. The raw data will be normalized with negative control decondensation value yielding the reportable value as % of the control.

Controls: In each test one negative control and one positive control will be run in parallel with the patient samples. The control specimen is a normal, SDD pre-tested specimen from a sperm bank, or a specimen obtained from an individual who has produced 4 or more ejaculates containing sperm that respond normally at the 15 min time point. This control serves as negative control when used in the complete SDD Test protocol. Patient positive controls that have been identified as being abnormal in routine testing of patient samples sent to Repromedix for analysis in the SDD Test are frozen and used as the positive control.

Outcome Evaluation:

All the results will be statistically evaluated.

Correlation between SDAD and the pregnancy success in the first (any) treatment selected.

Correlation between SDAD Test scores and pregnancy completion: Evaluation of the cut off value for the SDAD Test that best predicts pregnancy failure, (statistical PPV, NPV, specificity, sensitivity and accuracy).

Comparison between the correlations of SDAD Test scores and pregnancy results obtained within the same specimen used for ART versus those performed on a separate specimen.

Association between the SDAD Test, and environmental/drug/smoking exposures.

Construction of an improved decision-making paradigm involving the use of the SDAD Test results.

Statistical Analysis:

ANOVA, single factor without replication method and paired t tests (for consequential relations), will be used to compare each of the two test results (SDD, SPA) between patients with successful pregnancy and those with failed pregnancy. P value of <0.05 will be considered statistically significant. If data distribution deviates from normal distribution, or if the variances of the groups under analysis are dramatically different, rank tests (Mann Whitney Utest) would be performed. All data will be presented as mean±SEM.

Determination of the best cutoff value predicting pregnancy failure or specific pregnancy outcomes will be done by ROC analysis. Specificity, Sensitivity, PPV and NPV as well as Accuracy will be evaluated using the selected cutoff values.

In such as paired analysis will be performed.

Conclusion: This study demonstrates that the SDAD Test is highly predictive of ICSI outcome (See Table 2).

EXAMPLE 5 Use of Sperm DNA Decondensation (SDD™) Test in Predicting Fertility Benefit from a Varicocelectomy

A diagnostic test is needed for determining infertile males who can benefit from a varicocelectomy (VX). This example demonstrates that the SDD Test has this capacity, as well as utility in monitoring improvement of sperm post-surgery to determine when a couple should resume ART attempts at pregnancy. The SDD Test assesses the decondensation human sperm activation event (nuclear swelling) by incubating permeabilized human sperm in frog egg extract and observing how a patient's sperm decondensed relative to a normal fertile male's sperm. Any score less than 80% of the control is considered abnormal. The following Groups/Parameters were tested:

Individuals with abnormal SDD Test scores and with a varicocele(s) confirmed by a urologist can benefit from a varicocelectomy, and

SDD Test scores beginning 3 months post-surgery, individuals with substantial improvement will have a higher live birth outcome either by natural conception, or the use of ART.

Retrospective, single center study.

Materials and Methods:

Using medical records from 1 fertility practice, a retrospective chart review was performed on men sent to a urologist for varicocele evaluation because of an abnormal SDD Test score.

Results:

Nineteen males with abnormal SDD Test scores agreed to go to a urologist for further evaluation. Twelve of the 19 males were found to have varicocele(s). Nine males chose to have a VX, 3 males opted NOT to have the surgery. Of the 9 males who had the VX, 7 had repeat SDD Tests performed 3-5 months post-surgery. In all cases, substantial improvements in their SDD Test scores occurred. Two males had spontaneous conceptions (SC) and did not have a repeat SDD Test. One SC resulted in a live birth, the other spontaneously aborted at 6 weeks. Six of the 9 males with improved repeat scores fathered children either by natural conception (1) or by ART (3 singletons, 2 twins). One of the 7 males had a negative IVF. Thus, in the group who opted for the VX, 7 of 9 (78%) fathered children. No children (0%) were fathered by the 3 males who refused the VX.

These retrospective results demonstrate that the SDD Test can identify men with varicocele(s) who will have a fertility benefit from a varicocelectomy (p=0.045; Fisher Exact Test). The SDD Test may also be a useful marker of improved fertility potential of sperm after a varicocelectomy.

EXAMPLE 6 Sperm DNA Decondensation (SDD) Test and Selection of Assisted Reproductive Technology Method

Sperm DNA integrity testing has been reported to predict failure of sperm with assisted reproductive technology (ART), regardless of the method of insemination. It has been reported that sperm DNA structure tests (SCSA/SDFA) can identify reduced chances for success in some ART methods, such as intrauterine insemination (IUI). However, these tests are not deemed useful for others, such as in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI). Similarly, the sperm DNA decondensation (SDD) test, a sperm function test, has previously been shown to predict failure in subsequent ART attempts, regardless of method. The goal of this example is to demonstrate that the SDD Test has a predictive capability in current IUI and IVF insemination protocols.

Methods:

A retrospective review was performed on every male with SDD testing within a single fertility practice. Outcomes were identified for the first IUI or IVF attempt subsequent to SDD testing. A successful ART attempt was defined as presence of fetal heart beat at 8-10 weeks gestation or live delivery. Outcome of IUI or IVF was correlated with SDD score. An abnormal SDD score was defined as less than 80% of the control and normal was defined as 80% of the control or higher. There were no exclusions for female other male factor etiology.

Results:

SDD scores and first IUI or IVF attempt outcome data was available for 58 males. Forty-three males had normal SDD scores and had a 22% success rate with IUI (N=23) and 35% with IVF (N=20) attempts. Fifteen males had abnormal SDD scores with 0% successful outcome with IUI (N=6) and IVF (N=9) attempts. The difference between the 28% success rate of SDD normal patients (N=43, IUI/IVF combined) and 0% with SDD abnormal patients (N=15, IUI/IVF combined)) was statistically significant using Fischer's exact test (P=0.0325) (Odds Ratio>5). An additional 18 patients underwent IVF with ICSI where 81% (N=16) of males with normal SDD scores had success and 100% (n=2) with an abnormal SDD had success, although there was not sufficient statistical power to conclude that the groups were different. There was no significant difference for the average age and FSH levels of female partners between SDD normal and SDD abnormal groups.

Based upon the present data analysis, the SDD will identify male patients with reduced chances of success with IUI and IVF who may benefit from earlier consideration of

EXAMPLE 7 Automation of Scoring the SDAD and SDD Tests

Optical LiveCell Array Technology.

A method that provides a novel 96 well plate with etched glass at the bottom of each well with a transparent array of micron-sized wells, with sperm analysis as one application is provided. This plate will be used in the practice of the present example to accommodate the running of 8 assays at the same time and then transfer stained, fixed sperm into 8 wells at a time speeding up the processing of the samples. The image analysis system can then be used to analyze 500 sperm per well without the need of a technician individually scoring each test in real time using phase contrast microscopy. This system is being used to provide a way to let the fixed stained sperm settle into the wells, such that both accelerated decondensation (5 min time point) and delayed decondensation (15 min time point) may be determined.

Fixation and staining has been optimized, as well as the time that the sperm must be put into the well to settle into the live cell array so that a maximum number of sperm that are individual and non-clumped can be analyzed by an image analysis system. The results achieved with this protocol yield results that closely match the phase contrast results for the 15 min time point (SDD Test) where a delay in decondensation is examined. Phase contrast results at the 5 min time point (SDAD Test) will be further examined to determine a match of data/analysis when examining accelerated decondensation.

Sperm Prep: same as in the phase contrast approach to scoring.

Egg Extract: same as in the phase contrast approach to scoring.

In a standard 96 well plate, transfer 50 ul of egg extract into each well using a pipetteman with 8 pipette tips that fit into the 8 wells in each row of the 96 well plate, i.e., run 8 assays at a time. Add 4 ul of sperm at a concentration of 25,000 sperm per ul and mix well. Start the timer. After a 5 min incubation a 25 ul aliquot of the extract/sperm cocktail is placed in the next 8 wells of the 96 well plate, and 1 ul of Hoescht 33258 stain (1 ul of stock Hoescht 33258 stain in 1 ml of water; 1:1,000 dilution) is added to each well and fixation solution is added in5 μl aliquots per well (5 times) mixing the mixture in the well after each addition of fixative. The fixation solution is made by mixing a EM Grade paraformaldehyde, 16% w/v, Distilled Water, 100% v/v mix 1:1 with Phosphate Buffered Saline (PBS). The 50 ul of fixed stained sperm is then transferred into the 8 wells of the 96 well plate with the microwells at the bottom, into which the sperm will settle.

At the 10 min time point, 1 μl of 10% EtOH is added to the extract sperm incubation mixture. After incubating the sperm in the egg extract for 17 minutes, 1 ul of Hoescht 33258 stain (described above) is added to each of the 8 wells, and the fixation process is performed as described above. The 50 μl of fixed stained sperm is then transferred into the 8 wells of the 96 well plate with the microwells at the bottom, into which the sperm will settle. This process will be repeated for groups of 8 patient samples until the 96 well plate is filled. The plate will then be placed into a refrigerator for analysis the next day.

EXAMPLE 8 Manual Scoring of the Sperm DNA Accelerated Decondensation (SDAD) Test, and the Sperm DNA Decondensation (SDD™) Test Using Phase Contrast Microscopy

For both the SDAD, and SDD Tests:

Semen: aliquots will be kept in the refrigerator at 2-8° C., until shipped cold by packing with a coolpak provided with shipping supplies. Specimens must be sent no later than 9 days after sample collection, and must be shipped on Monday, Tuesday, Wednesday or Thursday so they will not arrive on the weekend. The samples will be analyzed in the SDAD and/or the SDD Tests within 14 days of sample collection. Any sample arriving such that the sample can not be examined within the 14 day QC window will be rejected, and another sample requested.

Egg Extracts Female Xenopus Laevis Oocyte positive frogs are maintained and the frog egg extracts produced as described in the attached Brown et. al. papers (1992, 1995). After preparing a frog egg extract Lot using a minimum of 15 frogs to provide eggs, the extract is immediately snap, deep-frozen as 30 ul drops in liquid N₂ (LN) that are transferred into cryo-vials that are kept in LN until thawed for use in performing the SDAD and SDD Tests. This frog egg extract will be used to induce the sperm DNA decondensation, mimicking the event after sperm entry into the oocyte.

The SDAD and SDD tests: About two million sperm per sample are washed and permeablized as described in the Brown et. al. papers (1992, 1995). After four extensive washes with special buffers, sperm are treated with a buffer containing dithiotreitol (DTT). The DTT-treated sperm are then incubated with frog egg extract to induce sperm DNA decondensation.

SDAD: After a 5 min incubation in egg extract, an aliquot of the sperm/egg extract mixture is placed on a glass slide and a cover slip is placed on top of the mixture. Approximately 50-75 sperm are scored in real time during a 5 min period of time, and the percentage of fully decondensed sperm is determined using phase contrast microscopy. The percentage of sperm fully decondensed is recorded. The raw data is normalized with a negative control (normal male as described below) decondensation value yielding the reportable value that is the percentage of the control sperm that have fully decondensed at 5 min. Any value less than 120% of the control is considered normal. Greater than 120% of the control is considered abnormal, and patients with such scores have a poor chance of having a successful ICSI attempt at pregnancy. This value is based on the results shown in FIG. 8.

SDD: After a 15 min incubation in egg extract, an aliquot of the sperm/egg extract mixture is placed on a glass slide and a cover slip is placed on top of the mixture. Approximately 50-75 sperm are scored in real time during a 5 minute period of time, and the percentage of fully decondensed sperm is determined using phase contrast microscopy. The percentage of sperm fully decondensed is recorded. The raw data is normalized with a negative control (normal male as described below) decondensation value yielding the reportable value that is the percentage of the control sperm that have fully decondensed at 15 minutes. Any score less than 80% of the control is considered an abnormal score. Such individuals have a poor chance of success in TUT or IVF-ET attempts at pregnancy. However, an abnormal SDD Test score cannot predict whether a patient will be successful in an ICSI attempt at pregnancy.

For both the SDAD and SDD Tests, in each test, one negative control and one positive control are run in parallel with the patient study samples. The negative control specimen is from a male who has produced 3 ejaculates that have normal decondensation at 15 min (typically 96±2 percent SD). Frozen donor sperm from a sperm bank can also be used, but the preferred negative control sample is an ejaculate kept at 4° C. for up to a month and used whenever needed during its 1 month shelf life. This control serves as a negative control when used in performing both the SDAD and SDD Test protocol. Positive controls (sperm that fails in the SDD Test) are routinely identified. Aliquots of these samples will be maintained for use as our positive control. The same protocol will be followed for the SDAD Test when it is offered commercially.

EXAMPLE 9 Automated Scoring Process Data

The present example demonstrates the clinical relationship of results obtained using the Phase Contrast and the Fourescent Image Analyses methods in using the SDAD assay. The present example also describes the equipment and presents clinical data in the form of captured images, and demonstrates the strong correlation, and hence predictive clinical value, between the Phase Contrast and Fourescent Image Analysis as tools in monitoring and screening a human sperm sample.

Hardware and Software:

MetaMorph Premier Acquisition system including workstation and hardware control capabilities for motoized microscope components, stage, camera, shutters as well as additional capabilities that can be used for future hardware integration.

Premier Off-line with LiveCell Array analysis drop-in for image processing. Hardware integration.

CoolSnap HQII scientific-grade digital camera and driver.

Motorized XY stage for Olympus IX71 microscope with controller, joystick and universal sample holder. Focus drive system to motorize control of IX71 microscope's fine focus.

Prior NanoScanZ 200 um Piezo Stage System. Includes sample holders for slides, dishes and microplates and DAQ Board For Piezo drive integration.

Uniblitz shutters for transmitted and fluorescent light paths with controller.

A 96 well plate with etched glass at the bottom of each well with a transparent array of micron-sized wells, with sperm analysis via image analysis software and hardware as one application.

A IX71 Olympus Inverted microscope with the filter system and a lwd 40× objective for fluorescence analysis of Hoescht 33258 stained sperm.

TABLE 3 SDD TEST: % fully decondensed sperm DNA at 15 min Phase Contrast Flourescence Image Sample Real time Analysis Auto Focus 1 93.3 90 2 57.8 63 3 96.4 95.2 4 44.8 38.9 5 83.3 76.6 6 92.3 93.2 7 100 100 8 94.4 93.9 Slope: 1.0147 Correlation coefficient: 0.9831 Intercept: −2.66

The automated scoring approach yields the same results as the laborious manual scoring approach described in Example 8 (See Table 3).

EXAMPLE 10 Automation of the Processing of the Sperm for Use in the SDAD and SDD Tests

This procedure will replace the manual protocol as described above. All the test steps will be done automatically by a liquid handling robotics from the first exposure of sperm to any reagent in the test itself to the fixation, so that no operator's action will be necessary. At the end of this process, the operator could transfer the plate to the refrigerator or to the automated scoring system described above.

Automated Performance of the Test Procedure:

The present example describes the protocol performed by the liquid handling robotics that mimics the manual operation. The present example also describes the equipment needed for this procedure, and provides a comparison between a test result run manually and one run on the automated system, on the same specimen(s). The equipment is basically a liquid handling robotics that can handle the following functions:

-   -   Sample cells from a mixture of sperm at a pre-set concentration         (eg. ten million sperm per milliliter), and set in an empty         plate in which the assay is to be conducted (the assay plate).     -   Aliquot other solutions from a stock test tube, and delivering         them to the assay plate.     -   Mix the sperm after any reagent addition, and before any aliquot         removal from the assay plate.     -   Maintain the assay plate and the reagents' vessels in various         desired temperature(s).

Mimicking the various steps in the manual process can then be done as in the example protocol as follows. However, this procedure is one example, but an automated approach is not limited to using the same concentrations, incubation times or steps as below. Thus, some steps may be performed in a different order, or perhaps eliminated altogether dependent on the particular conditions, etc., under consideration when running a particular sample batch, or dependent upon what materials are most readily available or convenient.

-   -   Aliquot a small volume of sperm suspension at a preset         concentration in NIM;     -   Add a small volume of concentrated Lysolecithin         (permeabilization) and mix Wait 5 minutes;     -   Add a high concentration of BSA to scavenge the Lysolecithin,         and mix;     -   Move to 4° C.;     -   Add a small volume of highly concentrated DTT;     -   Incubate one hr at 4° C.;     -   Add diluting/washing buffer (XEIM) and mix;     -   Withdraw a certain volume;     -   Add similar volume of XEIM to replace the withdrawn volume, and         mix;     -   Repeat the previous step to bring down the total cell numbers to         be similar to the earlier developed manual method;     -   Add ice cold frog egg extract, and mixincubate for 5 min (SDAD)         or 15 min (SDD);     -   Add Hoechst 33258 fluorescent dye in a few microliter volume, to         stain DNA;     -   Add Para formaldehyde fixative in several rounds of addition and         mixing; and     -   Mix well and transfer entire volume to bottom-etched scoring         plate.

BIBLIOGRAPHY

The following references are specifically incorporated herein in their entirety by reference:

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1. A sperm DNA accelerated decondensation test capable of assessing male fertility comprising: monitoring test sperm decondensation in a frog egg extract for a period of 5 minutes to 10 minutes, wherein a quantifiable difference between a test sperm decondensation and a known fertile male sperm decondensation after a period of 5 to 10 minutes provides an assessment of male fertility.
 2. The method of claim 1, wherein the frog egg extract is a Xenopus laevies frog egg extract. 